Generation and Recovery of &amp;beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

2017

Biomater. Sci.

Self Cite

Stiffening of the extracellular matrix is a hallmark in cancer progression, embryonic development, and wound healing. To mimic this dynamic process, our work explored orthogonal enzymatic reactions capable of modulating the properties of poly(ethylene glycol) (PEG)-peptide hydrogels. A hepta-mutant bacterial transpeptidase sortase A (SrtA) was used to ligate two PEG-peptide macromers (i.e., PEG-YLPRTG and NH-GGGG-PEG) into a primary hydrogel network. The hydrogels were dynamically stiffened using mushroom tyrosinase (MT), which oxidized tyrosine residues into di-tyrosine and led to increased matrix stiffness. After confirming the expression and enhanced catalytic activity of SrtA, we investigated the cytocompatibility of the enzymatic reaction with a mouse insulinoma cell line, MIN6. In addition, we altered peptide substrate concentrations and evaluated their influence on primary hydrogel network properties and MT-triggered stiffening. Using a pancreatic cancer cell line, COLO-357, the effect of MT-triggered stiffening on spheroid formation was investigated. We found that cell spheroids formed in hydrogels that were exposed to MT were significantly smaller than spheroids formed without MT incubation, suggesting that matrix stiffening played a crucial role in the sizes of cancer cell spheroids. Through utilizing highly specific and orthogonal enzymatic reactions, this hydrogel platform permits rapid and mild in situ cell encapsulation, as well as dynamic control of matrix stiffness for investigating the role of matrix stiffening on cell fate processes.

Section: Cytocompatibility Of Srta7m-crosslinked Hydrogelssupporting

confidence: 87%

Orthogonal enzymatic reactions for rapid crosslinking and dynamic tuning of PEG–peptide hydrogels

Arkenberg

2017

Biomater. Sci.

Self Cite

“…Our group has evaluated the cytocompatibility of PEG-based thiol-norbornene hydrogel using a radical-sensitive pancreatic β-cell line, MIN6, and concluded that the orthogonal thiol-ene ‘photo-click’ reaction is more cytocompatible than chain-growth PEGDA hydrogels at equivalent reactive macromer functionality. 37, 80 The inherent degradability of PEG-norbornene macromer (due to ester bond hydrolysis) also has been shown to increase proliferation and spreading of hMSCs. 41 Roberts and Bryant encapsulated bovine chondrocytes in chain-growth PEGDA and step-growth thiol-norbornene hydrogels and showed that the later promoted hyaline-like cartilage production with positive staining for aggrecan and collagen II, especially when the cell-laden hydrogels were cultured under mechanical loading.…”

Section: Thiol-norbornene Hydrogels In Tissue Engineering Applicatmentioning

confidence: 99%

Thiol–norbornene photoclick hydrogels for tissue engineering applications

J of Applied Polymer Sci

Han

2014

Self Cite

193

214

Thiol-norbornene (thiol-ene) photo-click hydrogels have emerged as a diverse material system for tissue engineering applications. These hydrogels are cross-linked through light mediated orthogonal reactions between multi-functional norbornene-modified macromers (e.g., poly(ethylene glycol), hyaluronic acid, gelatin) and sulfhydryl-containing linkers (e.g., dithiothreitol, PEG-dithiol, bis-cysteine peptides) using low concentration of photoinitiator. The gelation of thiol-norbornene hydrogels can be initiated by long-wave UV light or visible light without additional co-initiator or co-monomer. The cross-linking and degradation behaviors of thiol-norbornene hydrogels are controlled through material selections, whereas the biophysical and biochemical properties of the gels are easily and independently tuned owing to the orthogonal reactivity between norbornene and thiol moieties. Uniquely, the cross-linking of step-growth thiol-norbornene hydrogels is not oxygen-inhibited, therefore the gelation is much faster and highly cytocompatible compared with chain-growth polymerized hydrogels using similar gelation conditions. These hydrogels have been prepared as tunable substrates for 2D cell culture, as microgels or bulk gels for affinity-based or protease-sensitive drug delivery, and as scaffolds for 3D cell culture. Reports from different laboratories have demonstrated the broad utility of thiol-norbornene hydrogels in tissue engineering and regenerative medicine applications, including valvular and vascular tissue engineering, liver and pancreas-related tissue engineering, neural regeneration, musculoskeletal (bone and cartilage) tissue regeneration, stem cell culture and differentiation, as well as cancer cell biology. This article provides an up-to-date overview on thiol-norbornene hydrogel cross-linking and degradation mechanisms, tunable material properties, as well as the use of thiol-norbornene hydrogels in drug delivery and tissue engineering applications.

“…In contrast, MIN6 cells encapsulated in chain‐growth PEGDA hydrogels did not survive at low cell density . When appropriate peptide substrates (e.g., CGGY↓C, where arrow indicates chymotrypsin cleavage site) were used as thiol‐ene gel crosslinker, cell spheroids generated in situ were rapidly recovered via enzyme‐mediated gel erosion . In a separate study using experimental investigation and mathematical modeling, our laboratory has shown that thiol‐ene gels formed by multi‐arm PEG‐ester‐norbornene and dithiol‐containing linkers were susceptible to base‐catalyzed hydrolytic degradation .…”

Section: Introductionmentioning

confidence: 99%

The Influence of Matrix Degradation and Functionality on Cell Survival and Morphogenesis inPEG‐Based Hydrogels

Raza

Macromolecular Bioscience

2013

Self Cite

Poly(ethylene glycol) (PEG) hydrogels formed by thiol-norbornene photo-click reaction have been used in a variety of biomedical applications. The mild and rapid thiol-norbornene reaction permits the generation of versatile biomimetic extracellular matrices for studying cell behaviors in 3D. Thiol-norbornene PEG-based hydrogels can be rendered biodegradable if appropriate macromers and cross-linkers are employed. Previous studies have elucidated, experimentally and mathematically, in vitro degradation behaviors of these hydrogels. However, the highly tunable thiol-ene gel degradability and the influence of different forms of gel degradation on promoting cell survival and morphogenesis in 3D have not been fully evaluated. Toward this end, two norbornene-functionalized PEG macromers, namely PEG-tetra-ester-norbornene (PEG4eNB) and PEG-tetra-amide-norbornene (PEG4aNB), were synthesized to render the resulting hydrogels with different hydrolytic degradability. Di-thiol containing likers, such as dithiothreitol or bis-cysteine containing peptides, were utilized to control proteolytic degradability of the gels. The influence of thiol-ene gel degradability on cell survival and morphogenesis in 3D was assessed using human mesenchymal stem cells (hMSCs) and pancreatic MIN6 β-cells. The results showed that initial cell viability could be negatively affected in highly cross-linked thiol-norbornene hydrogels. In addition, when cells were encapsulated in thiol-ene gels lacking cell-adhesive motifs, their survival and proliferation were promoted in more hydrolytically labile hydrogels. Finally, the degree of 3D cell spreading in encapsulated hMSCs was enhanced when the matrices were immobilized with cell-adhesive motifs and were allowed to degrade both proteolytically and hydrolytically.